Crystallization of Intact Receptor Tyrosine Kinases

描述（由申请人提供）：受体酪氨酸激酶（RTKs），包括胰岛素和表皮生长因子（EGF）受体家族，介导许多动物组织中的细胞生长和分化。RTKs由细胞外配体结合区、单跨膜跨越区和细胞质激酶组成。RTKs的不适当激活导致细胞生长异常，功能失调的RTKs与许多人类癌症的发生和严重程度有关，并成为许多抗癌药物的靶点：例如，在20-30%的人类乳腺癌中发现了EGF受体同源物HER2的过表达，并与更具有侵袭性的肿瘤和更差的预后相关，抗HER2单克隆抗体赫赛汀已被证明是治疗这些癌症的有效药物。我的实验室最近开发了一种方法来生产高水平的可溶性富含半胱氨酸的糖蛋白，其中包括大多数rtk的细胞外区域，以适合x射线晶体学分析的形式。这项工作已经导致了EGF受体HER2、HER2与Herceptin Fab复合物以及EGF受体同源物HER3的细胞外区域的原子分辨率结构。这些结构为配体结合如何产生信号提供了许多见解，并激发了抗癌药物设计的新方法。然而，对于配体结合产生的信号是如何在细胞膜上传递的，人们知之甚少。完整受体的原子分辨率结构是解决这一问题的必要条件。确定这些结构的一个主要障碍是以结晶形式表达和纯化足够数量的完整受体。我们的第一个目标是将我们开发的可溶性富含半胱氨酸糖蛋白的表达方法应用于完整受体的表达和纯化。这些方法将普遍适用于所有细胞表面蛋白的结构研究。我们的第二个目标是将这些方法应用于EGF和胰岛素受体家族的过表达成员。我们的第三个目标是纯化和表征这些受体。我们的最终目标是生产这些受体单独或与配体络合的衍射质量晶体。

DESCRIPTION (provided by applicant): Receptor tyrosine kinases (RTKs), which include the insulin and epidermal growth factor (EGF) receptor families, mediate cell growth and differentiation in many animal tissues. RTKs consist of an extracellular ligand binding region, a single transmembrane spanning region, and a cytoplasmic kinase. Inappropriate activation of RTKs results in abnormal cell growth, and dysfunctional RTKs have been implicated in the genesis and severity of many human cancers and become the target of many anticancer drugs: For example, overexpression of the EGF receptor homolog HER2 is found in 20-30% of human breast cancers and correlates with more aggressive tumors and a poorer prognosis, and an anti-HER2 monoclonal antibody, Herceptin, has proven an effective treatment for these cancers. My laboratory has recently developed an approach to producing high levels of soluble cysteine-rich glycoproteins, which includes the extracellular regions of most RTKs, in forms suitable for X-ray crystallographic analysis. This work has led to atomic resolution structures of extracellular regions of the EGF receptor, HER2, HER2 complexed with the Herceptin Fab, and the EGF receptor homolog HER3. These structures have provided much insight into how ligand binding generates signals and inspired new approaches to anticancer drug design. Little insight has been gained, however, into how the signal produced by ligand binding is transduced across the cell membrane. Atomic resolution structures of intact receptors are needed to solve this problem. A major roadblock to determination of these structures is the expression and purification of sufficient amounts of intact receptors in crystallizable form. Our first aim is to adapt the expression methods that we have developed for soluble cysteine-rich glycoproteins to the expression and purification of intact receptors. These methods will be generally applicable to structural studies of all cell surface proteins. Our second aim is to apply these methods to overexpress members of the EGF and insulin receptor families. Our third aim is to purify and characterize these receptors. Our final aim is to produce diffraction-quality crystals of these receptors both alone and complexed with ligand.